Binding of Aldolase and Glyceraldehyde-3-Phosphate Dehydrogenase to the Cytoplasmic Tails of Plasmodium falciparum Merozoite Duffy Binding-Like and Reticulocyte Homology Ligands

ABSTRACTInvasion of erythrocytes byPlasmodium falciparumrequires a connection between the cytoplasmic tail of the parasite’s ligands for its erythrocyte receptors and the actin-myosin motor of the parasite. For the thromobospondin-related anonymous protein (TRAP) ligand onPlasmodiumsporozoites, aldolase forms this connection and requires tryptophan and negatively charged amino acids in the ligand’s cytoplasmic tail. Because of the importance of the Duffy binding-like (DBL) and the reticulocyte homology (RH) ligand families in erythrocyte binding and merozoite invasion, we characterized the ability of their cytoplasmic tails to bind aldolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), both of which bind actin. We tested the binding of the cytoplasmic peptides of the two ligand families to aldolase and GAPDH. Only the cytoplasmic peptides of some RH ligands showed strong binding to aldolase, and the binding depended on the presence of an aromatic amino acid (phenylalanine or tyrosine), rather than tryptophan, in the context of negatively charged amino acids. The binding was confirmed by surface plasmon resonance analysis and was found to represent affinity similar to that seen with TRAP. An X-ray crystal structure of aldolase at 2.5 Å in the presence of RH2b peptide suggested that the binding site location was near the TRAP-binding site. GAPDH bound to some of the cytoplasmic tails of certain RH and DBL ligands in an aromatic amino acid-dependent manner. Thus, the connection betweenPlasmodiummerozoite ligands and erythrocyte receptors and the actin motor can be achieved through the activity of either aldolase or GAPDH by mechanisms that do not require tryptophan but, rather, other aromatic amino acids.IMPORTANCEThe invasion of thePlasmodiummerozoite into erythrocytes is a critical element in malaria pathogenesis. It is important to understand the molecular details of this process, as this machinery can be a target for both vaccine and drug development. InPlasmodiumsporozoites andToxoplasmatachyzoites, invasion involves a glycolytic enzyme aldolase, linking the cytoplasmic tail domains of the parasite ligands to the actin-myosin motor that drives invasion. This binding requires a tryptophan that cannot be replaced by other aromatic residues. Here we show that aldolase binds the cytoplasmic tails of someP. falciparummerozoite erythrocyte-binding ligands but that the binding involves aromatic residues other than tryptophan. The biological relevance of aldolase binding to cytoplasmic tails of parasite ligands in invasion is demonstrated by our observation that RH2b but not RH2a binds to aldolase and, as previously shown, that RH2b but not RH2a is required forP. falciparuminvasion of erythrocytes.

Download Full-text

Histidine Degradation via an Aminotransferase Increases the Nutritional Flexibility of Candida glabrata

Eukaryotic Cell ◽

10.1128/ec.00072-14 ◽

2014 ◽

Vol 13 (6) ◽

pp. 758-765 ◽

Cited By ~ 13

Author(s):

Sascha Brunke ◽

Katja Seider ◽

Martin Ernst Richter ◽

Sibylle Bremer-Streck ◽

Shruthi Ramachandra ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Aromatic Amino Acid ◽

Candida Glabrata ◽

Genomic Structure ◽

Aromatic Amino Acids ◽

Sole Source ◽

Upstream Region ◽

Content Type ◽

Aromatic Amino Acid Aminotransferase

ABSTRACTThe ability to acquire nutrients during infections is an important attribute in microbial pathogenesis. Amino acids are a valuable source of nitrogen if they can be degraded by the infecting organism. In this work, we analyzed histidine utilization in the fungal pathogen of humansCandida glabrata. Hemiascomycete fungi, likeC. glabrataorSaccharomyces cerevisiae, possess no gene coding for a histidine ammonia-lyase, which catalyzes the first step of a major histidine degradation pathway in most other organisms. We show thatC. glabratainstead initializes histidine degradation via the aromatic amino acid aminotransferase Aro8. AlthoughARO8is also present inS. cerevisiaeand is induced by extracellular histidine, the yeast cannot use histidine as its sole nitrogen source, possibly due to growth inhibition by a downstream degradation product. Furthermore,C. glabratarelies only on Aro8 for phenylalanine and tryptophan utilization, sinceARO8, but not its homologueARO9, was transcriptionally activated in the presence of these amino acids. Accordingly, anARO9deletion had no effect on growth with aromatic amino acids. In contrast, inS. cerevisiae,ARO9is strongly induced by tryptophan and is known to support growth on aromatic amino acids. Differences in the genomic structure of theARO9gene betweenC. glabrataandS. cerevisiaeindicate a possible disruption in the regulatory upstream region. Thus, we show that, in contrast toS. cerevisiae,C. glabratahas adapted to use histidine as a sole source of nitrogen and that the aromatic amino acid aminotransferase Aro8, but not Aro9, is the enzyme required for this process.

Download Full-text

Structure-Activity Relationship of Synthetic Variants of the Milk-Derived Antimicrobial Peptide αs2-Casein f(183–207)

Applied and Environmental Microbiology ◽

10.1128/aem.01394-13 ◽

2013 ◽

Vol 79 (17) ◽

pp. 5179-5185 ◽

Cited By ~ 18

Author(s):

Avelino Alvarez-Ordóñez ◽

Máire Begley ◽

Tanya Clifford ◽

Thérèse Deasy ◽

Kiera Considine ◽

...

Keyword(s):

Amino Acids ◽

Biological Activity ◽

Amino Acid ◽

Antimicrobial Peptide ◽

Peptide Sequence ◽

Content Type ◽

Charged Amino Acids ◽

Food Borne ◽

Relationship Of ◽

Positively Charged

ABSTRACTTemplate-based studies on antimicrobial peptide (AMP) derivatives obtained through manipulation of the amino acid sequence are helpful to identify properties or residues that are important for biological activity. The present study sheds light on the importance of specific amino acids of the milk-derived αs2-casein f(183–207) peptide to its antibacterial activity against the food-borne pathogensListeria monocytogenesandCronobacter sakazakii. Trimming of the peptide revealed that residues at the C-terminal end of the peptide are important for activity. Removal of the last 5 amino acids at the C-terminal end and replacement of the Arg at position 23 of the peptide sequence by an Ala residue significantly decreased activity. These findings suggest that Arg23 is very important for optimal activity of the peptide. Substitution of the also positively charged Lys residues at positions 15 and 17 of the αs2-casein f(183–207) peptide also caused a significant reduction of the effectiveness againstC. sakazakii, which points toward the importance of the positive charge of the peptide for its biological activity. Indeed, simultaneous replacement of various positively charged amino acids was linked to a loss of bactericidal activity. On the other hand, replacement of Pro residues at positions 14 and 20 resulted in a significantly increased antibacterial potency, and hydrophobic end tagging of αs2-casein f(193–203) and αs2-casein f(197–207) peptides with multiple Trp or Phe residues significantly increased their potency againstL. monocytogenes. Finally, the effect of pH (4.5 to 7.4), temperature (4°C to 37°C), and addition of sodium and calcium salts (1% to 3%) on the activity of the 15-amino-acid αs2-casein f(193–207) peptide was also determined, and its biological activity was shown to be completely abolished in high-saline environments.

Download Full-text

Molecular Determinants of the Cofactor Specificity of Ribitol Dehydrogenase, a Short-Chain Dehydrogenase/Reductase

Applied and Environmental Microbiology ◽

10.1128/aem.07751-11 ◽

2012 ◽

Vol 78 (9) ◽

pp. 3079-3086 ◽

Cited By ~ 17

Author(s):

Hee-Jung Moon ◽

Manish Kumar Tiwari ◽

Ranjitha Singh ◽

Yun Chan Kang ◽

Jung-Kul Lee

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Binding Pocket ◽

Site Directed Mutagenesis ◽

Titration Calorimetry ◽

Content Type ◽

Cofactor Specificity ◽

Charged Amino Acids ◽

Molecular Determinants ◽

Ribitol Dehydrogenase

ABSTRACTRibitol dehydrogenase fromZymomonas mobilis(ZmRDH) catalyzes the conversion of ribitol tod-ribulose and concomitantly reduces NAD(P)+to NAD(P)H. A systematic approach involving an initial sequence alignment-based residue screening, followed by a homology model-based screening and site-directed mutagenesis of the screened residues, was used to study the molecular determinants of the cofactor specificity of ZmRDH. A homologous conserved amino acid, Ser156, in the substrate-binding pocket of the wild-type ZmRDH was identified as an important residue affecting the cofactor specificity of ZmRDH. Further insights into the function of the Ser156 residue were obtained by substituting it with other hydrophobic nonpolar or polar amino acids. Substituting Ser156 with the negatively charged amino acids (Asp and Glu) altered the cofactor specificity of ZmRDH toward NAD+(S156D, [kcat/Km,NAD]/[kcat/Km,NADP] = 10.9, whereKm,NADis theKmfor NAD+andKm,NADPis theKmfor NADP+). In contrast, the mutants containing positively charged amino acids (His, Lys, or Arg) at position 156 showed a higher efficiency with NADP+as the cofactor (S156H, [kcat/Km,NAD]/[kcat/Km,NADP] = 0.11). These data, in addition to those of molecular dynamics and isothermal titration calorimetry studies, suggest that the cofactor specificity of ZmRDH can be modulated by manipulating the amino acid residue at position 156.

Download Full-text

The TK0271 Protein Activates Transcription of Aromatic Amino Acid Biosynthesis Genes in the Hyperthermophilic Archaeon Thermococcus kodakarensis

mBio ◽

10.1128/mbio.01213-19 ◽

2019 ◽

Vol 10 (5) ◽

Author(s):

Yasuyuki Yamamoto ◽

Tamotsu Kanai ◽

Tsuyoshi Kaneseki ◽

Haruyuki Atomi

Keyword(s):

Amino Acids ◽

Transcriptional Regulation ◽

Amino Acid ◽

Aromatic Amino Acid ◽

Amino Acid Biosynthesis ◽

Acid Biosynthesis ◽

Content Type ◽

Hyperthermophilic Archaeon ◽

Thermococcus Kodakarensis ◽

Aromatic Amino Acid Biosynthesis

ABSTRACT TrpY from Methanothermobacter thermautotrophicus is a regulator that inhibits transcription of the Trp biosynthesis (trp) operon. Here, we show that the TrpY homolog in Thermococcus kodakarensis is not involved in such regulation. There are 87 genes on the T. kodakarensis genome predicted to encode transcriptional regulators (TRs). By screening for TRs that specifically bind to the promoter of the trp operon of T. kodakarensis, we identified TK0271. The gene resides in the aro operon, responsible for the biosynthesis of chorismate, a precursor for Trp, Tyr, and Phe. TK0271 was expressed in Escherichia coli, and the protein, here designated Tar (Thermococcales aromatic amino acid regulator), was purified. Tar specifically bound to the trp promoter with a dissociation constant (Kd) value of approximately 5 nM. Tar also bound to the promoters of the Tyr/Phe biosynthesis (tyr-phe) and aro operons. The protein recognized a palindromic sequence (TGGACA-N8-TGTCCA) conserved in these promoters. In vitro transcription assays indicated that Tar activates transcription from all three promoters. We cultivated T. kodakarensis in amino acid-based medium and found that transcript levels of the trp, tyr-phe, and aro operons increased in the absence of Trp, Tyr, or Phe. We further constructed a TK0271 gene disruption strain (ΔTK0271). Growth of ΔTK0271 was similar to that of the host strain in medium including Trp, Tyr, and Phe but was significantly impaired in the absence of any one of these amino acids. The results suggest that Tar is responsible for the transcriptional activation of aromatic amino acid biosynthesis genes in T. kodakarensis. IMPORTANCE The mechanisms of transcriptional regulation in archaea are still poorly understood. In this study, we identified a transcriptional regulator in the hyperthermophilic archaeon Thermococcus kodakarensis that activates the transcription of three operons involved in the biosynthesis of aromatic amino acids. The study represents one of only a few that identifies a regulator in Archaea that activates transcription. The results also imply that transcriptional regulation of genes with the same function is carried out by diverse mechanisms in the archaea, depending on the lineage.

Download Full-text

Protein Targeting to the Parasitophorous Vacuole Membrane of Plasmodium falciparum

Eukaryotic Cell ◽

10.1128/ec.00008-11 ◽

2011 ◽

Vol 10 (6) ◽

pp. 744-752 ◽

Cited By ~ 17

Author(s):

Saliha Eksi ◽

Kim C. Williamson

Keyword(s):

Amino Acids ◽

Plasmodium Falciparum ◽

Amino Acid ◽

Fluorescent Protein ◽

Transmembrane Domain ◽

Signal Sequence ◽

Parasitophorous Vacuole ◽

Reporter Protein ◽

Content Type ◽

Gfp Reporter

ABSTRACTRed blood cell (RBC) invasion and parasitophorous vacuole (PV) formation byPlasmodium falciparumare critical for the development and pathogenesis of malaria, a continuing global health problem. Expansion of the PV membrane (PVM) during growth is orchestrated by the parasite. This is particularly important in mature RBCs, which lack internal organelles and no longer actively synthesize membranes. Pfs16, a 16-kDa integral PVM protein expressed by gametocytes, was chosen as a model for studying the trafficking of material from the parasite across the PV space to the PVM. The locations of Pfs16-green fluorescent protein (GFP) reporter proteins containing distinct regions of Pfs16 were tracked from RBC invasion to emergence. Inclusion of the 53 C-terminal amino acids (aa) of Pfs16 to a GFP reporter construct already containing the N-terminal secretory signal sequence was sufficient for targeting to and retention on the PVM. An amino acid motif identified in this region was also found in seven other known PVM proteins. Removal of the 11 C-terminal aa did not affect PVM targeting, but membrane retention was decreased. Additionally, during emergence from the PVM and RBC, native Pfs16 and the full-length Pfs16-GFP reporter protein were found to concentrate on the ends of the gametocyte. Capping was not observed in constructs lacking the amino acids between the N-terminal secretory signal sequence and the transmembrane domain, suggesting that this region, which is not required for PVM targeting, is involved in capping. This is the first report to define the amino acid domains required for targeting to theP. falciparumPVM.

Download Full-text

Tailoring a Global Iron Regulon to a Uropathogen

mBio ◽

10.1128/mbio.00351-20 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 1

Author(s):

Rajdeep Banerjee ◽

Erin Weisenhorn ◽

Kevin J. Schwartz ◽

Kevin S. Myers ◽

Jeremy D. Glasner ◽

...

Keyword(s):

Amino Acids ◽

Urinary Tract ◽

Amino Acid ◽

Regulatory Network ◽

Iron Homeostasis ◽

Iron Limitation ◽

Content Type ◽

E Coli ◽

General Stress ◽

K 12

ABSTRACT Pathogenicity islands and plasmids bear genes for pathogenesis of various Escherichia coli pathotypes. Although there is a basic understanding of the contribution of these virulence factors to disease, less is known about variation in regulatory networks in determining disease phenotypes. Here, we dissected a regulatory network directed by the conserved iron homeostasis regulator, ferric uptake regulator (Fur), in uropathogenic E. coli (UPEC) strain CFT073. Comparing anaerobic genome-scale Fur DNA binding with Fur-dependent transcript expression and protein levels of the uropathogen to that of commensal E. coli K-12 strain MG1655 showed that the Fur regulon of the core genome is conserved but also includes genes within the pathogenicity/genetic islands. Unexpectedly, regulons indicative of amino acid limitation and the general stress response were also indirectly activated in the uropathogen fur mutant, suggesting that induction of the Fur regulon increases amino acid demand. Using RpoS levels as a proxy, addition of amino acids mitigated the stress. In addition, iron chelation increased RpoS to the same levels as in the fur mutant. The increased amino acid demand of the fur mutant or iron chelated cells was exacerbated by aerobic conditions, which could be partly explained by the O2-dependent synthesis of the siderophore aerobactin, encoded by an operon within a pathogenicity island. Taken together, these data suggest that in the iron-poor environment of the urinary tract, amino acid availability could play a role in the proliferation of this uropathogen, particularly if there is sufficient O2 to produce aerobactin. IMPORTANCE Host iron restriction is a common mechanism for limiting the growth of pathogens. We compared the regulatory network controlled by Fur in uropathogenic E. coli (UPEC) to that of nonpathogenic E. coli K-12 to uncover strategies that pathogenic bacteria use to overcome iron limitation. Although iron homeostasis functions were regulated by Fur in the uropathogen as expected, a surprising finding was the activation of the stringent and general stress responses in the uropathogen fur mutant, which was rescued by amino acid addition. This coordinated global response could be important in controlling growth and survival under nutrient-limiting conditions and during transitions from the nutrient-rich environment of the lower gastrointestinal (GI) tract to the more restrictive environment of the urinary tract. The coupling of the response of iron limitation to increased demand for amino acids could be a critical attribute that sets UPEC apart from other E. coli pathotypes.

Download Full-text

The Carboxy-Terminal Region ofFlavobacterium johnsoniaeSprB Facilitates Its Secretion by the Type IX Secretion System and Propulsion by the Gliding Motility Machinery

Journal of Bacteriology ◽

10.1128/jb.00218-19 ◽

2019 ◽

Vol 201 (19) ◽

Cited By ~ 5

Author(s):

Surashree S. Kulkarni ◽

Joseph J. Johnston ◽

Yongtao Zhu ◽

Zachary T. Hying ◽

Mark J. McBride

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Cell Surface ◽

Type A ◽

Secretion System ◽

Gliding Motility ◽

Foreign Protein ◽

Type B ◽

Content Type ◽

Type Ix Secretion System

ABSTRACTFlavobacterium johnsoniaeSprB moves rapidly along the cell surface, resulting in gliding motility. SprB secretion requires the type IX secretion system (T9SS). Proteins secreted by the T9SS typically have conserved C-terminal domains (CTDs) belonging to the type A CTD or type B CTD family. Attachment of 70- to 100-amino-acid type A CTDs to a foreign protein allows its secretion. Type B CTDs are common but have received little attention. Secretion of the foreign protein superfolder green fluorescent protein (sfGFP) fused to regions spanning the SprB type B CTD (sfGFP-CTDSprB) was analyzed. CTDs of 218 amino acids or longer resulted in secretion of sfGFP, whereas a 149-amino-acid region did not. Some sfGFP was secreted in soluble form, whereas the rest was attached on the cell surface. Surface-attached sfGFP was rapidly propelled along the cell, suggesting productive interaction with the motility machinery. This did not result in rapid cell movement, which apparently requires additional regions of SprB. Secretion of sfGFP-CTDSprBrequired coexpression withsprF, which lies downstream ofsprB. SprF is similar in sequence toPorphyromonas gingivalisPorP. MostF. johnsoniaegenes encoding proteins with type B CTDs lie immediately upstream ofporP/sprF-like genes. sfGFP was fused to the type B CTD from one such protein (Fjoh_3952). This resulted in secretion of sfGFP only when it was coexpressed with its cognate PorP/SprF-like protein. These results highlight the need for extended regions of type B CTDs and for coexpression with the appropriate PorP/SprF-like protein for efficient secretion and cell surface localization of cargo proteins.IMPORTANCETheF. johnsoniaegliding motility adhesin SprB is delivered to the cell surface by the type IX secretion system (T9SS) and is rapidly propelled along the cell by the motility machinery. How this 6,497-amino-acid protein interacts with the secretion and motility machines is not known. Fusion of the C-terminal 218 amino acids of SprB to a foreign cargo protein resulted in its secretion, attachment to the cell surface, and rapid movement by the motility machinery. Efficient secretion of SprB required coexpression with the outer membrane protein SprF. Secreted proteins that have sequence similarity to SprB in their C-terminal regions are common in the phylumBacteroidetesand may have roles in adhesion, motility, and virulence.

Download Full-text

Human transferrin receptor internalization is partially dependent upon an aromatic amino acid on the cytoplasmic domain.

Cell Regulation ◽

10.1091/mbc.1.4.369 ◽

1990 ◽

Vol 1 (4) ◽

pp. 369-377 ◽

Cited By ~ 77

Author(s):

T E McGraw ◽

F R Maxfield

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Rate Constant ◽

Transferrin Receptor ◽

Aromatic Amino Acid ◽

Opposite Polarity ◽

Receptor Internalization ◽

Aromatic Side Chain ◽

Human Transferrin ◽

Human Transferrin Receptor

The objective of this work is to identify the elements of the human transferrin receptor that are involved in receptor internalization, intracellular sorting, and recycling. We have found that an aromatic side chain at position 20 on the cytoplasmic portion of the human transferrin receptor is required for efficient internalization. The wild-type human transferrin receptor has a tyrosine at this position. Replacement of the Tyr-20 with an aromatic amino acid does not alter the rate constant of internalization, whereas substitution with the nonaromatic amino acids serine, leucine, or cysteine reduces the internalization rate constant approximately three-fold. These results are consistent with similar studies of other receptor systems that have also documented the requirement for a tyrosine in rapid internalization. The amino terminus of the transferrin receptor is cytoplasmic, with the tyrosine 41 amino acids from the membrane. These two features distinguish the transferrin receptor from the other membrane proteins for which the role of tyrosine in internalization has been examined, because these proteins have the opposite polarity with respect to the membrane and because the tyrosines are located closer to the membrane (within 25 amino acids). The externalization rate for the recycling of the transferrin receptor is not altered by any of these substitutions, demonstrating that the aromatic amino acid internalization signal is not required for the efficient exocytosis of internalized receptor.

Download Full-text

Bacterially Expressed Full-Length Recombinant Plasmodium falciparum RH5 Protein Binds Erythrocytes and Elicits Potent Strain-Transcending Parasite-Neutralizing Antibodies

Infection and Immunity ◽

10.1128/iai.00970-13 ◽

2013 ◽

Vol 82 (1) ◽

pp. 152-164 ◽

Cited By ~ 54

Author(s):

K. Sony Reddy ◽

Alok K. Pandey ◽

Hina Singh ◽

Tajali Sahar ◽

Amlabu Emmanuel ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Neutralizing Antibodies ◽

Malaria Vaccine ◽

Expression System ◽

Full Length ◽

Blood Stage ◽

Parasite Protein ◽

Content Type ◽

Erythrocyte Binding ◽

Blood Stage Malaria

ABSTRACTPlasmodium falciparumreticulocyte binding-like homologous protein 5 (PfRH5) is an essential merozoite ligand that binds with its erythrocyte receptor, basigin. PfRH5 is an attractive malaria vaccine candidate, as it is expressed by a wide number ofP. falciparumstrains, cannot be genetically disrupted, and exhibits limited sequence polymorphisms. Viral vector-induced PfRH5 antibodies potently inhibited erythrocyte invasion. However, it has been a challenge to generate full-length recombinant PfRH5 in a bacterial-cell-based expression system. In this study, we have produced full-length recombinant PfRH5 inEscherichia colithat exhibits specific erythrocyte binding similar to that of the native PfRH5 parasite protein and also, importantly, elicits potent invasion-inhibitory antibodies against a number ofP. falciparumstrains. Antibasigin antibodies blocked the erythrocyte binding of both native and recombinant PfRH5, further confirming that they bind with basigin. We have thus successfully produced full-length PfRH5 as a functionally active erythrocyte binding recombinant protein with a conformational integrity that mimics that of the native parasite protein and elicits potent strain-transcending parasite-neutralizing antibodies.P. falciparumhas the capability to develop immune escape mechanisms, and thus, blood-stage malaria vaccines that target multiple antigens or pathways may prove to be highly efficacious. In this regard, antibody combinations targeting PfRH5 and other key merozoite antigens produced potent additive inhibition against multiple worldwideP. falciparumstrains. PfRH5 was immunogenic when immunized with other antigens, eliciting potent invasion-inhibitory antibody responses with no immune interference. Our results strongly support the development of PfRH5 as a component of a combination blood-stage malaria vaccine.

Download Full-text